HILIC-NMR: Toward the Identification of Individual Molecular

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HILIC-NMR: Toward the Identification of Individual Molecular Components in Dissolved Organic Matter Gwen C. Woods,† Myrna J. Simpson,† Philip J. Koerner,‡ Antonia Napoli,‡ and Andr
e J. Simpson†,* † ‡

Department of Chemistry, University of Toronto, Ontario, Canada, MIC1A4 Phenomenex Inc, 411 Madrid Avenue, Torrance, California 90501, United States

bS Supporting Information ABSTRACT: This article presents research targeted toward the isolation and detection of unique molecular structures from what is believed to be the world’s most complex organic mixture: dissolved organic matter (DOM). Hydrophilic interaction chromatography (HILIC) was used to separate Suwannee River DOM (SRDOM) into 80 fractions, simplified to the extent that detection with nuclear magnetic resonance spectroscopy (NMR) results in many sharp signals that are indicative of individual compounds, some of which are identifiable with multidimensional NMR. Parallel factor analysis (PARAFAC) of fluorescence excitation
emission matrices (EEMs) was additionally employed on HILIC-simplified fractions to further confirm the effectiveness of the HILIC separations as well as draw insight into how structural characteristics relate to DOM fluorescence signals. Findings suggest that material believed to be derived from both cyclic and linear terpenoids was dominant in the most hydrophobic fractions as were the majority of the fluorescence signals, whereas hydrophilic material was highly correlated with carbohydrate-type structures as well as high contributions from amino acid fluorescence. NMR spectra of DOM, typically featureless mounds, are substantially more detailed with HILIC-simplified fractions to the point where hundreds of signals are present and 2D NMR correlations permit significant structural identifications.

’ INTRODUCTION Dissolved organic matter (DOM) is ubiquitous throughout the world’s aquatic ecosystems and is thought to be one of the most complex natural substances on Earth.1 Despite research dating back more than a century,2 DOM has proven difficult to characterize at the molecular-level; it has been estimated that a mere 1
10% of DOM can be resolved into specific chemical structures3 using conventional analytical approaches. Fourier transform ion cyclotron resonance-mass spectrometry (FTICRMS) and nuclear magnetic resonance (NMR) spectroscopy have been cited as among the most promising techniques for obtaining the molecular resolution necessary for DOM elucidation.1,4 The application of FTICR-MS with DOM has the potential for extreme resolution and research in recent years has enabled the identification of 1000s of molecular formulas.1,4,5 The limitation, however, is that distinct structural formulas are not elucidated and become progressively more difficult to ascertain with increasing molecular mass. Even with the resolving power of FTICR-MS, constituents of ∼600 Da result in 15 possible molecular formulas which in turn represent on the order of 100 000
1 000 000s of possible structural formulas.5 Multidimensional, advanced NMR techniques have the capacity to solve bonding and structural inquiries and have enabled researchers to identify plausible types of structures present in marine and freshwater DOM such as carboxyl-rich alicyclic molecules r 2011 American Chemical Society

(CRAM)6 and material derived from linear terpenoids (MDLT);7 both structural classifications likely originate from terpenoids and have been estimated to account for as much as 75% of DOM.7 NMR analysis of DOM, in turn, is limited in that severe signal overlap from the many constituents present hinders extensive structural assignments. Further developments with both these analytical techniques as well as novel research approaches are therefore necessary to reach the resolution necessary for the assignment of substantial structural formulas. A lack of sufficient chromatographic resolution of DOM has been cited as a reason for the shortage of molecular information8 and substantial improvements with chromatography is likely critical to obtain the homogeneity necessary for substantial identifications via both NMR and FTICR-MS. This material is believed to be a complex mixture ranging in polarity that is aggregated into large, supramolecular structures,9
11 and thus chromatographic separations have proven to be challenging. High-performance liquid chromatography (HPLC), for example, typically results in very few resolved signals 4,11
13 indicating that the 1000s of DOM compounds present are not substantially Received: October 19, 2010 Accepted: March 28, 2011 Revised: March 26, 2011 Published: April 06, 2011 3880

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Environmental Science & Technology separated. The HPLC techniques used to date, however, have primarily focused on high-performance size exclusion chromatography (HPSEC) or reverse phase HPLC (RP-HPLC). Surface adsorption chromatography, such as RP-HPLC, is best accomplished if analytes have varying affinities for the stationary and mobile phases. If constituents present have little affinity for the stationary phase they will elute quickly and be poorly resolved. Hydrophilic interaction chromatography (HILIC) is similar to normal-phase HPLC with a polar stationary phase but utilizes partial aqueous mobile phase. HILIC separations are appropriate for polar retention,14 whereas in complex environmental samples the less polar constituents will interact much less with the polar stationary phase and subsequently elute with the earliest material. Retention of constituents are achieved via multiple mechanisms, the most influential being analyte partitioning between organic-rich eluent and a thin water layer near the polar stationary surface. Charge interaction, hydrogen-bonding, dipole
dipole interactions, and hydrophobic effects further affect analyte selectivity.14,15 HILIC is well suited for HPLC separations of highly oxidized environmental samples and has the added advantage of permitting high sample loads (via partitioning mechanisms).14 To the best of our knowledge, this research is the first application of HILIC with natural organic matter and we here report the separation of IHSS Suwannee River DOM (SRDOM) into simplified fractions prior to offline NMR detection. It is important to note that HILIC is based on hydrophilic, or polar, interactions and is hence ideally suited for the separation of polar material, which are prolific in the aquatic environment and which are challenging to separate using more conventional chromatographic approaches. Excitation
emission matrices (EEMs) were further collected on fractions and deconvoluted with parallel factor analysis (PARAFAC) to draw insight into fluorescence characteristics. Fluorescence techniques are rapid, highly sensitive, widely employed, and useful for the identification of source material such as terrestrially derived fluorescence versus microbially derived amino acid fluorescence.16
18 The two techniques are complementary in that they target different types of information: NMR overall structures present and fluorescence the minor constituents present that fluoresce most strongly. Understanding how structural information is linked to fluorescence signals is therefore useful and applied here to further characterize the separation of DOM with HILIC. PARAFAC has generally been used for the characterization of bulk DOM samples, but is considered ideal for DOM samples collected along a gradient 19 and is applied here to a gradient of simplified fractions. The objectives of this current research thus include: A) to assess the effectiveness of HILIC as a novel method applied to the separation of DOM and to utilize more than one analytical method to determine the variability and subsequent success of the material separated, B) to determine how structural information from NMR varies with polarity, C) to determine how PARAFAC components vary with polarity, and finally D) to link the results of the widely used PARAFAC methods with the less accessible NMR-derived structural information in hopes of drawing insight into how structural groups relate to DOM fluorescence phenomena.

’ EXPERIMENTAL SECTION HILIC Separation. HILIC was used to separate SRDOM into 80 fractions that were repeatedly collected over 60 runs and

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lyophilized. HILIC separations were achieved on an Agilent 1200 series system with a diode array detector (DAD, Agilent model G1315B) monitored at 254, 280, and 320 nm, a fluorescence detector (FLD, Agilent model G1321A) monitored at λexc = 320 nm and λem = 430 nm, and an analytical fraction collector (Agilent model G1364C). A Phenomenex, LUNA column was used (4.6  15 mm, 3 μm particle size, comprised of silica derivatized with cross-linked diol functional groups, fitted with a prefilter and guard column). A complex gradient of acetonitrile/aqueous was used for the mobile phase. Further details of the separation and fraction collection are provided in the Supporting Information. NMR Analysis. HILIC fractions were analyzed on a Bruker AvanceTM 500 MHz spectrometer at 298 K with a 1H
13C-15N 1.7 mm microprobe fitted with an actively shielded z-gradient. Lyophilized fractions were prepared (2 mg per 50 μL) in D2O with 1% NaOD, sonicated to ensure complete dissolution, and injected into 1.7 mm microtubes. For most 1D experiments, 1024 scans were acquired with 16k time domain points and a recycle delay of 2s. Select samples were rerun with 32k time domain points to obtain spectra with high resolution (referred to here as high-resolution spectra). For nonhigh-resolution experiments, presaturation utilizing relaxation gradients and echoes (PURGE)20 was used to suppress the signal from water at ∼4.7 ppm. Because of improved peak shape when processed without line broadening, high-resolution experiments were acquired using composite pulse presaturation.21 Most 1D spectra were apodized with an exponential multiplication factor of 1 Hz; the high-resolution experiments were Fourier transformed directly without the use of a window function. All NMR spectra were processed using a zero filling factor of 2 and referenced externally to DSS (4,4-dimethyl-4-silapentane-1-sulfonic acid) at 0 ppm. 2D correlation spectroscopy with a 45° pulse (COSY45) spectra were obtained with 512 transients, with increments of 2048 and 256 for F2 and F1, respectively. Each spectrum was zero-filled by a factor of 2 and with 5500 Hz spectral widths. The recycle delay was 1s for all COSY45 experiments and the data were processed in absolute value mode. A range of additional NMR experiments including TOCSY, HSQC, HSQC-TOCSY, and HMBC were also collected and assignments were confirmed with these additional data sets. Experimental parameters along with discussion of the TOCSY and HSQC experiments may be found in the Supporting Information. Fluorescence Analysis. For fluorescence analyses, aliquots of 5 μL were taken from freshly prepared NMR samples (just prior to addition into NMR tubes) and diluted to ∼5 mg/L DOC with HPLC-grade water. All EEMs were collected on an Agilent 1200 series fluorescence detector (G1321A), equipped with a xenon flash lamp and an offline cuvette for EEMs acquisition. EEMs were collected using excitation from 230 to 450 nm (5 nm increments) to generate emission spectra from 280 to 550 nm (2 nm increments). Instrument corrections were accounted for following procedures from previous studies18,22 and are outlined in the Supporting Information along with procedures for the verification of the PARAFAC model. All samples were run in duplicate and found to be reproducible within a standard error of 0.99) for all chemical shifts between the proposed database structure and the resonances in DOM (detail provided in the Supporting Information). The confirmed structural assignments are presented in Figure 3 and are comprised of a variety of carboxyl, hydroxy and aliphatic acids. The additional detail provided by 2D NMR after fractionation is likely due to a combination of: 1) enhanced homogeneity, 2) reduced interaction within microenvironments giving rise to sharper signals, and 3) separation removed paramagnetics that enhance relaxation. Fluorescence Analysis. Validation of the PARAFAC model resulted in a 7-component system; Figure S4 of the Supporting Information illustrates these 7 components as well as an example of the modeling results of a HILIC fraction. Detailed discussion of these components may be found in the Supporting Information but in summation: 4 components were found to contain signals influenced by the addition of a variety of model quinones (Q1, Q2, Q3, Qb), one of these specifically to p-benzoquinone (Qb). The remaining 3 components are all attributed to amino acid fluorescence: a tyrosine component (TYR), and 2 tryptophan components (TRP1, TRP2). The 2 tryptophan signals are likely the result of the anisotropy of tryptophan fluorescence so that TRP1 results in most environments while TRP2 only occurs in association with very apolar constituents. Correlation data 3883

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Figure 3. 2D COSY45 NMR spectra of HILIC-simplified fraction (H09); left: zoomed region from 0.5 to 4.5 ppm, right: zoomed aromatic region (6.3
8.5 ppm). Assignments made from reference database (main text); (*) indicates assignment from previous work using a database of lignin components.29

indicate that all quinones (except Qb) and TRP2 are correlated with the most hydrophobic material while TYR and TRP1 are in turn most prominent in hydrophilic fractions (Supporting Information). Much like the NMR data, the PARAFAC components were found to vary with polarity with the exception of Qb, which was found more uniformly throughout all 80 fractions (more information in Supporting Information). The variation of the remaining components illustrates that the fractions were chemically distinct and this chemical distinction was further verified by two fractions that were dominated by single PARAFAC components. The 18th fraction (H18) was found to be predominantly Q1, quinone-influenced signal (80%), whereas the much more hydrophilic fraction (H69) was identifiable as tryptophan-derived (TRP1: 97%) (data not shown). The isolation of TRP1 is further illustrated in the chromatogram (part A of Figure 1). The arrow indicates the elution of H69 and a prominent, isolated peak is evident within this fraction. The dominance of single components within fractions demonstrates that the HILIC fractions are considerably more homogeneous than start material. Statistical Analysis of NMR and PARAFAC. The PCA loadings were examined for the combined NMR and PARAFAC data set to determine how fluorescence phenomena relate to the structures present throughout the 80 HILIC fractions. PC1, PC2, and PC3 account for 45.2%, 20.4%, and 8.7% of the data, respectively. To simplify the graphical presentation, the loadings and scores are plotted with just the PC1 and PC2 dimensions (65.6% of variance explained, no loss of information by omitting the third dimension) and are presented in Figure 4. The plots are used as a means of pattern matching to determine how the scores relate to the loadings. Points furthest from zero have the greatest impact on variance so that scores and loadings in approximate collocations are indicative of a link between samples and variables. The scores plot illustrates that the HILIC fractions wrap counterclockwise around the plot from the PC1(
)/PC2(
) to

the PC1(
)/PC2(þ) region from the least polar to most polar material. Across this arc in the loadings plot, the PARAFAC components follow in the order of Q3 f TRP2 f TYR f Q2 f Q1 f TRP1, whereas NMR groups follow from: MDLT f CRAM f arom f carb. Qb was not near any other loading and in a region of sparse scores. The proximity of Q3 and TRP2 with MDLT and in a region associated with the most apolar fractions suggests that material high in MDLT-type structures is responsible for the nonpolar tryptophan signal as well as Q3. TYR and Q2 are in turn clustered near NMR resonances within the CRAM region as well as shown to be associated with fractions ∼H14
H28 in the scores plot. Q1 is linked to midlate fractions as are resonances from the aromatic region. TRP1 is in turn associated with the most polar fractions as are resonances from the carbohydrate region. The overall trend would thus appear to be that quinone-influenced signals are linked to apolar constituents and largely associated with the resonances for CRAM and MDLT-type material. The tryptophan signals were found to occupy opposite regions of the PCA plot so that the polar TRP1 and nonpolar TRP2 are nicely associated with the polar and nonpolar scores, respectively. The PCA results provide evidence that tyrosine-type fluorescence is weighted in the PC1(þ)/ PC2(
) in association with the scores from more apolar fractions despite the tyrosine signal having a positive correlation with polarity (R2 = 0.55, p < 0.005). This finding suggests that the tyrosine signal is prominent in polar fractions but is also an important factor in more apolar material. The methods used to analyze the HILIC-separated fractions of SRDOM provide evidence that HILIC is an effective approach for DOM fractionation. 1D 1H NMR spectra are characterized by dozens of sharp signals, indicative of individual molecules, and such detail is not possible without substantial simplification of DOM. Major structural groups are shown to either increase or decrease with polarity such that PCA analysis demonstrates that there is a progression of material that differs slightly from fraction 3884

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Figure 4. PCA plots of the (A) loadings and (B) scores of the combined NMR and PARAFAC data set. The loadings for PARAFAC are highlighted in orange and labeled accordingly, circled regions are discussed in the main text.

to fraction and that apolar material is structurally very distinct from polar constituents. PARAFAC analysis of fluorescence signals further demonstrates variability across the polarity gradient with quinone-influenced signals most apolar and amino acid-type fluorescence most prominent in polar fractions. Fractions dominated by single fluorescence components further provide evidence for the homogeneity of the material. Finally, and perhaps most notably, the material is now sufficiently resolved within NMR spectra that discrete structural assignments can now be made with multidimensional experiments
a feat not readily accomplished on DOM samples. Such identifications can aid in determining the abundance of unknown molecules present in DOM as well as aid in understanding source material and processes that form DOM. This novel application of HILIC for DOM separation reveals that molecular-level elucidation is possible with NMR and that further development of these techniques and particularly further improvements in mobile/ stationary phase choices for DOM separation may prove indispensable to molecular-level identifications.

’ ASSOCIATED CONTENT

bS

Supporting Information. Additional information on the experimental as well as lignin identification with NMR. This material is available free of charge via the Internet at http://pubs. acs.org.

’ AUTHOR INFORMATION Corresponding Author

*Phone þ1 416 287 7547, fax þ1 416 287 7279, e-mail: andre. [email protected].

’ ACKNOWLEDGMENT The authors thank the Natural Science and Engineering Research Council of Canada (Discovery Grant, A.J.S) and the

International Polar Year (IPY) for providing funding. A.J.S. would further like to thank the government of Ontario for providing funding in the form of an Early Researcher Award.

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